Important knowledge about the properties of a sample can be acquired by investigating the lifetime of the excited states of the sample marked with one or more fluorescent dyes. In particular when multiple fluorescent dyes are used, with the aid of fluorescence lifetime imaging microscopy (FLIM), for example, it is possible to acquire knowledge about a sample region being investigated, for example its composition or environment. In cell biology, for example, the calcium concentration in a sample region can be inferred indirectly by measuring the lifetime of the fluorescent dyes.
FLIM is, in particular, a technique with which the various fluorescence decay times (caused e.g. by the use of various dyes or by different microenvironments) can be depicted as contrast in a microscopic image. Here the fluorescence decay times measured are depicted pixel by pixel, different colors being associated, for example, with pixels having different fluorescence decay times.
There are numerous methods for measuring the lifetime of the excited states of fluorescent dyes. Some of these methods are described in detail in chapters 4 and 5 of the textbook by Joseph R. Lakowicz, “Principles of Fluorescence Spectroscopy,” Kluwer Academic/Plenum Publishers, second ed., 1999. For example, it is possible to modulate the power output of the excitation light over time in order to infer, from the phase delay of the emitted light, the lifetime of the excited state.
It is also possible to excite the fluorescent dyes using short light pulses so that the time offset of the emitted light pulses can be measured electronically. DE 10 2004 017 956 A1, for example, discloses a microscope for investigating the lifetime of excited states in a sample, having at least one light source that generates excitation light and having at least one detector that receives the detected light proceeding from the sample. The microscope is characterized in that the light source contains a semiconductor laser that emits pulsed excitation light, an adjusting apparatus being provided for adjusting the pulse repetition rate to the specific lifetime properties of the sample.
The electronic system necessary for data evaluation in particular is offered commercially, often in the form of plug-in PC cards. A time-measuring card of this kind is, however, very complex and expensive.